Supplementary Information
F
F
H
H
H
Hindered
attack
O
SF
NR'
R
2
2
R
H
H
H
H
H
poor yield
trans-isomer
H
H
H
H
H
H
H
HH
R
H
H
O SF2NR'2
H
H F
R
H
H
Diaxial conformationof H-OH
H
R
enables dehydration
Fcis-isomer
H
H
H
predominant E2 product
Additional Scheme Explaination for poor yields in the fluorination of substituted cyclohexanols, adapted from Mange. 16
Synthesis of 4-fluorocyclohexane carboxylic acid
4-Fluorocyclohexanone
Method 1
HF-pyridine (0.86 mL) was added to a 4-hydroxycyclohexanone (103 mg, 0.91 mmol) contained
within a plastic vessel cooled to 0°C and stirred under nitrogen for two hours. The reaction was
quenched by the addition of ice cold water (1 mL) and extracted into ethyl acetate (3 x 10 mL). The
organic phase was dried over MgSO4, filtered and the solvent removed in vacuo. The product was
purified by flash chromatography on silica, eluting with a mixture of petroleum ether and ethyl
acetate (9:1) to give the title compound as a yellow transparent oil (26 mg, 0.22 mmol, 24 %).
Method 2
Diethylaminosulphur trifluoride (DAST) (0.35 mL, 2.65 mmol) in dichloromethane (20 mL), cooled
to –78°C was added via cannula to a solution of 4-hydroxycyclohexanone (300 mg, 2.63 mmol) in
dichloromethane (100 mL) previously cooled to -78°C and stirred under nitrogen. The solution was
then placed in an ice bath and left to stir at 0 °C. The reaction went to completion after one hour,
before water (30 mL) was added. The organic phase was separated, dried over MgSO4, filtered and
the solvent removed in vacuo. The product was purified by flash chromatography on silica, eluting
with a mixture of petroleum ether and ethyl acetate (9:1) to give the title compound as a yellow
transparent oil (54 mg, 0.47 mmol, 18 %):
δH (CDCl3) 1.85-2.10 (m, 2H), 2.26-2.37 (m, 4H), 2.53-2.72 (m, 2H), 4.99 (dm, 1H, JHF = 48 Hz), δc
(CDCl3) 29.5, 31.2 (d, 2C, 3JCF = 28.3 Hz), 36.2 (d, 2C, 4JCF = 6.1 Hz), 87.3 (d, JCF = 115.7 Hz),
300.0. δF (CDCl3) 187.0; MS (EI) m / z 117.1 (M+1); IR (neat film): max(cm-1) 1716 (C=O).
4-Fluorocyclohexancarbonitrile
Tosyl methyl isocyanide (55.5 mg, 0.28 mmol) in 1, 1-dimethoxyethane (DME) (0.7 mL) was added
to 4-fluorocyclohexanone (30 mg, 0.26 mmol) in DME (0.7 mL) stirring under nitrogen before
cooling to 0°C. Potassium tert-butoxide (58.1 mg, 0.52 mmol) in tert-butanol (0.8 mL) and DME
(0.4 mL) was added via cannula to the previous cooled mixture. The reaction was left to stir for 3.5
hours at room temperature until completion and ether (30 mL) added. The organic phase was washed
with aqueous NaHCO3 solution (10%, 2×10 mL), saturated aqueous NaCl solution (2 ×10 mL), dried
on MgSO4, and filtered. The solvents were removed in vacuo. Flash chromatography was not
employed as the product rapidly decomposed on silica. The crude product was dissolved in ether
(1mL), filtered through cotton wool and the filtrate evaporated in vacuo to give a 6 to 4 mixture of
the trans and cis isomer (29.5 mg, 0.23 mmol, 88%) as a light brown oil: δH (CDCl3) 1.52-1.85 (m,
4H), 1.90-2.05 (m, 4H), 2.54-2.65 (m, 0.6 H, CH-CN, trans), 2.72-2.82 (m 0.4 H, CH-CN, cis), 4.21
(dm, 0.6H, JHF = 48 Hz, trans), 4.27 (dm, 0.6H, JHF = 48 Hz, cis). δC (CDCl3) 24.71 (d, 4JCF = 4.9
Hz), 29.19 (d, 3JCF = 22.2 Hz), 29.711, 129.19 (d, JCF = 165.1 Hz), 152.164. δF (CDCl3) 182.4 (2s);
MS (EI) m/z =128.0 (M+1); IR (neat film): max(cm-1) 2233 (-CN).
4-Fluorocyclohexane carboxylic acid
A solution of aqueous sodium hydroxide (1M, 3.5 mL), and ethanol (0.35 mL) was added to 4fluorocyclohexancarbonitrile (40.5 mg, 0.32 mmol) in ethanol (0.36 mL), and heated for 4 hours in
an oil bath at 82°C. The ethanol was evaporated in vacuo. The aqueous phase was washed with
diethyl ether (10 mL), acidified to between pH 1 and 2 using concentrated hydrochloric acid,
extracted with diethyl ether (3×10 mL), the organic extracts combined, dried on sodium sulphate,
filtered and evaporated to give a 6 to 4 mixture of the trans and cis isomer (28 mg, 0.192 mmol,
60%) as a light brown resin: δH (CDCl3) 1.38-2.12 (m, 8H), 2.20-2.38 (m, 1H), 4.43 (dm, 0.4H, JHF
= 54 Hz, trans), 4.72 (dm, 0.6H, JHF = 48 Hz, cis); δC (MHz, CDCl3) 23.3 (d, 4JCF = 4.0 Hz, trans)
26.0 (d, 4JCF = 14.1 Hz, cis), 30.2 (d, 3JCF = 28.2, Hz, trans), 31.3 (d, 3JCF = 26.2 Hz, cis), 41.5
(trans), 41.7 (cis), 88.5 (d, JCF = 225.4 Hz, trans), 91.14 (d, JCF = 229.4 Hz, cis), 181.6 (trans),
181.7 (cis), δF (CDCl3) 174.7, 183.3; MS: (EI) m / z = 147.0 (M+1); IR (KBr pellet): max(cm-1)
2945 (OH), 1701 (=O).16
Extraction Procedure and LC-MS data
The fermentation broth (1 ml) was extracted with ethylacetate (1 ml). The organic phase was
evaporated to dryness, and the residue resuspended in methanol (0.2 ml). The resultant solution (50
μL) was used for analysis by LCMS. LCMS was performed on an integrated Agilent HP1100 HPLC
system in combination with a Bruker Daltonics Esquire 3000+ electrospray mass spectrometer
operating in positive ion mode. Chromatography was achieved over a Phenomenex Hyperclone
column (C18 BDS, 3u, 150 x 4.6 mm) eluting over 11 min at a flow rate of 1 mL/min with a linear
gradient from acetonitrile / water (40/60) to acetonitrile / water (80/20).
The feeding of cyclohexane carboxylic acid 3 yielded 39-deshydroxypre-rapamycin that
co-elutes with a previously generated sample reported by Gregory et al.11 RT = 8.5 min. [M+Na]+,
m/z = 848.5; [M-H]-, m/z = 824.4; UV, λmax = 277 nm (exhibiting the typical rapamycin triene
motif).
The feeding of 4-hydroxycyclohexane carboxylic acid 5 also yielded 39-deshydroxypre-rapamycin
the analytical data for this compound are identical to those detailed above.
Feeding of 3-hydroxycyclohexane carboxylic acid 6 led to the production of a peak eluting at 8.8
min distinct to that reported above but with identical MS and UV characteristics.
Feeding the 3-hydroxy-1ene-cyclohexane carboxylic acid 8 led to the production of a rapamycin
analogue, also eluting at 8.8 min again with identical MS and UV characteristics to the rapamycin
analogue generated through the feeding of compound 3.
Upon feeding compounds 7, 12 and 13 to the microorganism and extracting the culture media it was
not possible to detect any rapamycin.